Project II is designed to discover the molecular basis of CFTR-mediated restraint of ENaC activity in human ainway epithelia. CFTR-mediated chloride secrefion and and ENaC-mediated sodium absorption have been considered central to control of ain/vay surface liquid hydration, but the concept of reciprocal control of these opposing ion transport pathways is controversial. In some tissues where CFTR and ENaC are each expressed, their stimulafion is coordinated to achieve Na and Cl absorption. Indeed, that is the case in alveolar type 2 cells (AT2C), which are to be characterized in Project III ofthis PPG. However, in ainway epithelia, abundant evidence suggests that CFTR restrains ENaC function. We recently reported that CFTR and ENaC coimmunoprecipitate in normal ainway epithelia, and ENaC in CF bronchial epithelium undergoes more extensive cleavage than ENaC in normal. Because ENaC mediated Na+ absorpfion is acfivated by cleavage of ENaC extracellular domains, this finding is congruent with the nofion that CFTR restrains ENaC cleavage in normal bronchial epithelia. Project II proposes to identify the molecular basis of CFTR-ENaC associafions in ainway epithelia, which will be accomplished by biochemical assays, comparisons of CFTR and ENaC associated proteins in bronchial airway cells and AT2C, and molecular modeling of detected interactions (Aim 1). With this information. Project II will test two mechanistic hypotheses that describe inhibifion of ENaC cleavage. In Aim 2, we hypothesize that physical interacfion of the R-domain of CFTR with the cytosolic N-termini of ENaC inhibits ENaC proteolysis and activafion. This hypothesis is based on the novel observation that ENaC proteolysis is sfimulated by phosphoinositde-binding of its cytosolic Nterminal tails. In Aim 3, Project II will test the requirement for CFTR function in the regulation of ENaC cleavage, and will further test specifically how CFTR's control of ASL pH mediates CFTR-specific effects on ENaC proteolysis. Information from these aims will further our understanding of CFTR regulation of ENaC funcfion, specifically addressing the questions of why this regulation is cell specific, and yet when present, how it may be mediated by mulfiple mechanisms.